Huntington's disease (HD) is a late-onset neurodegenerative genetic disorder that produces uncontrolled physical movements, devastates intellectual faculties and triggers emotional disturbances. Those suffering from HD typically die within 10 years after symptoms surface and there is currently no cure. There is an emerging link between HD and clathrin-mediated endocytosis through Huntingtin-interacting protein 1 (HIP1). The purpose of this renewal application is to elucidate how the interaction between clathrin-coated vesicles and HIP1 (and HIP1R, R for related) is regulated in healthy cells to gain insights into the role of clathrin- mediated endocytosis in HD. A closely related purpose of this proposal is to find what structural determinants mediate the binding of HIP1 to Huntingtin-interacting protein 1-protein interactor (HIPPI), an accessory protein that modifies the activity of HIP1 in Huntington's disease. The first specific aim is to determine what destabilizes the opened region of HIP1 to understand if molecular flexibility in the coiled-coil of this protein is important for its function. Site-directed mutagenesis will target specific positions in the coiled-coil to see if the opened region of HIP1 can be stabilized. Deletion mapping studies will be performed to pinpoint the location of hard-wired structure in the opened region that was found to be highly resistant to temperatures that would inactivate other proteins in the body. The powerful methods of X-ray crystallography and Nuclear Magnetic Resonance (NMR) will be used to study the static and dynamic properties of the stable sub-structure unit to assess if this feature is significant for HIP1 function. The second specific aim is to determine if the binding dynamics of clathrin to HIP1 and HIP1R are regulated through molecular flexibility. First, the surfaces of HIP1 and HIP1R that make contact with the light chain subunit of clathrin will be fully characterized. Second, two binding models will be evaluated to determine if the binding determinants for clathrin light chain are spread between the two helices of dimeric HIP1 or if the determinants are contained entirely in one of the two helices. Third, a mutagenesis approach will determine if clathrin light chain and HIP proteins bind in a head-to-head or head-to-tail direction to deepen our understanding of how clathrin-coated vesicles build up in cells. The third specific aim is to finish the 3.7 crystal structure of the clathrin trimer domain to understand if this domain can interact productively with the N-terminus of HIP1. The fourth specific aim is to investigate what structural determinants control the formation of the HIPPI/HIP1 complex that triggers the caspase-3/caspase-8 apoptotic pathway in Huntington's disease. Planned mutagenesis and in vivo studies will focus on two highly charged regions in the opened region of HIP1. The sum total of the information that will flow from the proposed research will deepen our understanding of how HIP proteins function in clathrin-mediated endocytosis and how HIP1, specifically, can potentiate the progress of Huntington's disease.